Our laboratory is studying the 70-kDa class of heat shock proteins (Hsp70s and Hsc70s) which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes members of the DnaJ class of proteins act as necessary cofactors with the Hsc70 proteins, apparently presenting protein substrates to Hsc70. One of the processes where a DnaJ homolog is required for Hsc70 action is the uncoating of clathrin-coated vesicles. In studying the mechanism of uncoating, we discovered that the nerve-specific 100 kDa clathrin assembly protein, auxilin, is required for the uncoating process to occur, and we also showed that auxilin is a DnaJ homolog. We have now found that a 20 kDa recombinant fragment of auxilin that is no longer able to induce clathrin polymerization still acts as a cofactor with Hsc70 to cause uncoating, and we are planning to determine the structure of this recombinant fragment using NMR. In addition to these studies on auxilin we have studied the ATP dependent polymerization of Hsc70 by DnaJ homologs, an effect that may represent presentation of one Hsc70 to another by DnaJ homologs under conditions where substrates are not present. These studies suggest that DnaJ homologs not only induce Hsc70 to rapidly hydrolyze ATP but also transiently open up its substrate binding site. Our studies on the structure of the 20 kDa auxilin fragment both by itself and in the presence of Hsc70 and clathrin baskets should yield information on how DnaJ homologs accomplish these two effects. In our studies on the uncoating reaction, we have also made observations that are pertinant to the general mechanism of receptor mediated endocytosis. We found that Hsc70 can be bound to clathrin in two different ways. Immediately following uncoating, a stable clathrin-Hsc70-ADP complex forms from which bound Hsc70 dissociates very slowly. However, after ADP dissociates and ATP rebinds to this complex, even though the Hsc70 remains bound to the clathrin (thus preventing further uncoating) it is now in rapid equilibrium with free hsc70. Interestingly, this rapid equilibrium complex only forms in the presence of clathrin assembly proteins and once formed, it continues to be present in both ATP and ADP for several hours. Therefore, in nerve cells carrying out rapid endocytosis, it may act as a nucleating complex that does not polymerize in the cytosol but facilitates formation of clathrin-coated pits. In support of this view, we have now obtained evidence that this rapid equilibrium complex does indeed interact with stripped vesicle membranes causing transient rebinding of clathrin to the membranes in an ATP-dependent process.